6440
J. Agric. Food Chem. 2010, 58, 6440–6448 DOI:10.1021/jf100112x
Glycoprotein Expression in Human Milk during Lactation )
JOHN W. FROEHLICH,† ERIC D. DODDS,† MARIANA BARBOZA,† ERICA L. MCJIMPSEY,† RICHARD R. SEIPERT,† JIMI FRANCIS, HYUN JOO AN,† SAMARA FREEMAN,‡ J. BRUCE GERMAN,‡ AND CARLITO B. LEBRILLA*,†,§ Department of Chemistry, ‡Department of Food Science and Technology, and §School of Medicine, University of California, Davis, California 95616, and Department of Biochemistry, University of Nevada, Reno, Nevada 89557 )
†
While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor. KEYWORDS: Breast milk; glycoprotein; lactation; milk; post-translational modification
INTRODUCTION
Breast milk is the remarkable product of the mammary gland emerging from evolutionary pressure as the exclusive food for the growing infant (1-11). Because it serves as the sole source of nutrition in early life, milk has been widely studied to better understand the quantities and bioavailabilities of essential nutrients. Milk not only delivers nutrients but also provides the infant with a number of physiological advantages such as enhanced immunity (9, 12) and the development and maintenance of a complex microflora in the gastrointestinal tract (11, 13-15). In light of these varied and extensive benefits, it is critical to study the structure-function relationship of different components in milk and how they confer advantages to the infant. Proteins make up a major component of human milk. Proteins as a whole are the fourth most abundant component of human milk after lactose, lipids, and oligosaccharides. Total protein concentrations are typically in the 10-20 mg/mL range and have been shown to decrease during the lactation period, reaching a minimum after approximately 1 week. This change is also correlated with a drastic increase in total milk production and a change in overall protein composition. A more detailed study of these dynamic behaviors is especially interesting because milk proteins and their degradation products have been associated with a number of important biological activities. These include immunomodulatory (16), antibacterial (17), and pathogenbinding (18) activities. The rapidly developing infant requires a dynamic and complex source of nutrients, and the overall health, development, and *To whom correspondence should be addressed. Tel: 1-530-7526364. Fax: 1-530-754-5609. E-mail:
[email protected].
pubs.acs.org/JAFC
Published on Web 04/23/2010
protection of the infant are enhanced by a variety of bioactivities also present in milk. To understand these activities, research in this group has examined the behavior of important components in human milk during the course of the lactation period. We have recently quantified human milk oligosaccharides (HMOs) during the lactation period in these samples and have found them to remain relatively constant for all components (19) during lactation. One proposed role of HMOs is as a prebiotic (15, 20), and the constant production of oligosaccharides is consistent with that role. Variation of protein expression is similarly important given the many roles of proteins in the infant diet. While thorough conventional proteomic studies have been performed on both the whey (4, 21) and the milk fat globule membrane (22, 23) fractions of human milk, there are very few studies that address any variation in post-translational modifications. Even fewer studies address milk protein glycosylation, although there have been a few notable exceptions. In 2002, Charlwood et al. (23) described the N-glycosylation of four abundant milk fat globule membrane proteins. This study interrogated samples from a wide range of lactation stages, yet reported neither any temporal variations in glycan abundance that are apparent in this study nor variations in protein abundances. While there have been studies establishing the variation in both total protein quantity and protein composition in human milk, little progress has been made in determining any qualitative or quantitative variations in glycosylation of the major milk glycoproteins. Protein glycosylation is one of the most commonly occurring post-translational modifications and directly affects protein structure, function, and recognition. Protein glycosylation is particularly relevant to milk, as it has been shown to have
© 2010 American Chemical Society
Article direct implications in pathogen binding (8), infant development, and proteolytic susceptibility (24). In this work, glycosylation of specific milk proteins is shown to vary during lactation. The work presented here both outlines a strategy for rapidly determining gross protein glycosylation profiles over time and demonstrates that there are dynamically glycosylated glycoproteins in human milk. Dynamic protein glycosylation suggests many interesting biological roles for individual proteins. An extensive review has been published on the potential roles of protein-linked glycosylation (25). As changes in protein glycosylation can significantly affect protein function, stability, and structure (26-28), tracking these changes could facilitate the discovery of additional bioactive species in milk. The degradation products from these compounds may be equally as important as the intact protein, as they have been shown to perform various biological functions both in vivo and in vitro (8, 12, 29, 30). Whereas glycosylation will greatly influence the function, identity, and quantities of these degradation products, a more thorough understanding of temporal variations of protein glycosylation is clearly necessary. As the neonate’s intestinal microflora, immune system, and digestive systems change the most rapidly during the first few days of life, understanding changes in protein expression and glycosylation are critically important. MATERIALS AND METHODS Materials. Organic solvents [all high-performance liquid chromatography (HPLC) grade or higher] were purchased from Burdick and Jackson. Sequencing-grade trypsin (modified by reductive methylation to reduce autolysis) was purchased from Promega. Bisacrylamide gels, Bradford reagent, and Coomassie brilliant blue G-250 were purchased from Bio-Rad. All water used was 18 MΩ deionized water. Pro-Q emerald 300 was purchased from Invitrogen. Glycerol-free Peptide: N-glycosidase F (PNGaseF) was purchased from New England Biolabs. Porous graphitized carbon cartridges were obtained from Glygen. All other reagents were purchased from Sigma-Aldrich. Samples. Human milk samples were obtained from four healthy women. Overall, samples from days 1, 2, 5, 10, 15, 16, 17, and 30, 31, or 32 of lactation were interrogated in this study, although none of the individuals provided milk for all time points. Quantitative comparisons were made with a minimum of three samples for each time point, and days 15, 16, and 17 were grouped for this comparison as were days 30, 31, and 32. All milk samples were manually expressed and immediately frozen. Samples were then transferred to a -80 °C freezer within 3 h and stored there until analysis. Protein Extraction. One half milliliter of raw milk was centrifuged at 4 °C for 30 min, and the fat and cellular layers were removed. Residual lipids were removed by the method of Wessel and Flugge (31). Briefly, three volumes (1.5 mL) of 2:1 chloroform/methanol was added, the solution was agitated, and the supernatant was retained. An ethanol precipitation of protein from the supernatant was performed overnight at 4 °C by adding 5 mL of HPLC-grade ethanol, and following centrifugation, the supernatant was removed. Precipitates were resuspended in 50 mM ammonium bicarbonate buffer (pH 7.5), and protein quantities were determined by the Bradford method. The precipitated protein was stored at -20 °C until analysis. Soduim Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). An aliquot containing 10 μg of protein was mixed 1:1 (v/v) with Laemmli sample buffer containing 350 mM dithiothreitol (DTT) (32) and was denatured and reduced by 2 min of heating at 95 °C. SDS-PAGE separations were achieved with discontinuous gradient 10-20% bisacrylamide gels and constant 20 mA running conditions. Following separations, gels were washed four times for 10 min with water. Subsequent staining with Coomassie brilliant blue and the glycoproteinspecific Pro-Q Emerald 300 was performed in parallel on duplicate gels according to the manufacturer’s instructions. Gels were scanned using an HP ScanJet 4890 scanner (in the case of Coomassie-stained gels) or a BioRad transilluminating scanner (in the case of Pro-Q Emerald-stained gels).
J. Agric. Food Chem., Vol. 58, No. 10, 2010
6441
In-Gel Trypsin Digestion. Selected gel bands were thoroughly washed with deionized water and gently agitated for a total of 1 h. Gel pieces were cut into 1 mm wide cubes and dried in a vacuum centrifuge. Washed, dehydrated gel pieces were incubated with 10 mM DTT at 55 °C for 1 h and 55 mM iodoacetamide (IoAA) at room temperature in the dark for 45 min. Gel pieces were then washed with 100 mM ammonium bicarbonate with gentle agitation for 10 min and briefly dehydrated with acetonitrile. This wash step was repeated twice, and gel pieces were dried. Proteins were then digested with 0.5 μg of trypsin in 100 mM ammonium bicarbonate at 37 °C for 16 h. Following digestion, peptides were extracted with acetonitrile, water, and 50% ethanol and dried in a vacuum centrifuge. Samples were reconstituted with deionized water, desalted using a zip-tip C18 microtip, and eluted into 5 μL of 50% acetonitrile. One microliter was used for subsequent matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry (MS) analysis, as described below. Peptide Mass Fingerprinting Analysis with MALDI-MS. Tryptic peptides were analyzed on an IonSpec ProMALDI 7.0 T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) equipped with an external MALDI source and capable of external ion accumulation with vibrational cooling. All spectra were internally calibrated using the InCAS technique (33) and a mixture of standard peptides to increase mass accuracy. The peptides used were Bradykinin fragment peptides 1-7, angiotensin II, P14R, human adrenocorticotropic hormone fragment peptides 18-39, and oxidized B chain of bovine insulin. Protonated, monoisotopic masses for the calibrant peptides were m/z 757.39915, 1046.54179, 1533.85765, 2465.19833, and 3494.65077, respectively. Spectra were deisotoped using the IonSpec PeakHunter software, and the monoisotopic mass lists were then relieved of known contaminants and nonpeptide masses using the Mass Sieve approach (34). MASCOT Database Searching. Processed mass lists were submitted to MASCOT (35) for protein identification. Carbamidomethylation of cysteine residues was selected as a fixed modification. No variable modifications were included in the searches. Peptide mass tolerances were set to 10 ppm. All proteins were identified above the 95% confidence interval (CI), and mass errors for all assigned peptides were less than 5 ppm. Each band identified only one protein (discounting splice variants) at that confidence level. Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/ MS) Analyses. A total of 11 samples were examined via LC-MS/MS. One hundred micrograms of extracted milk proteins was dried and solubilized with 60 μL of 8 M urea. Samples were then reduced with DTT and alkylated with IoAA. After dilution in 180 μL of water, an overnight digestion with trypsin was performed. Peptides were then concentrated and desalted using C18 zip-tip before LC separation and online MS/MS. A nanoLC-2D system (Eksigent, Dublin, CA) coupled with an LTQ ion trap mass spectrometer (Thermo Finnigan) was used with a homemade fritless reverse phase microcapillary column (75 μm 180 mm; packed with Magic C18AQ, 3 μm 100 A˚: Michrom Bio Resources) and vented column configuration. Digested samples were transferred from the autosampler to the online trap column (0.15 mm 20 mm; packed with Magic C18AQ, 3 μm 100 A˚) and desalted. Peptides were eluted from the trap and separated on the capillary column using a reverse-phase gradient at a flow rate of 300 nL/min and directly electrosprayed into the mass spectrometer. A cycle of one MS survey scan followed by 10 MS/MS scans was repeatedly acquired over the LC gradient. Dynamic exclusion for 1 min duration was utilized. Buffers were 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). A 107 min gradient (2-40% B for 95 min, followed by 40-80% B for 12 min) was used. Protein identification based on LC-MS/MS was performed using X! Tandem with a fragment ion mass tolerance of 0.40 Da and a parent ion tolerance of 1.8 Da. IoAA derivatization of cysteine was specified as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, sulfonation of methionine, tryptophan oxidation to formylkynurenin of tryptophan, acetylation of lysine and the N-terminus, and phosphorylation of serine, threonine, and tyrosine were specified as variable modifications. Scaffold software was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide
6442
J. Agric. Food Chem., Vol. 58, No. 10, 2010
Froehlich et al.
Prophet algorithm (36). Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet (37). In-Gel Deglycosylation and MALDI FTICR-MS Analysis. To verify changes in lactoferrin glycosylation, 10 μg of milk protein corresponding to lactation days 2 and 5 was reduced, alkylated, and loaded on to 10% polyacrylamide gels (86 mm 68 mm 1 mm). After separation, protein visualization was performed using Bio-Safe Coomassie blue staining. Lactoferrin gel bands were excised and destained with 5% methanol-7% acetic acid in water and frozen overnight. After extensive washing with acetonitrile and 50 mM NH4HCO3, glycans were released using 750 units of PNGaseF at 37 C for 16 h. Released glycans were extracted from gel pieces by washing them with 200 μL of water, followed by 200 μL of acetonitrile, with a 10 min sonication step following each addition. Supernatants obtained in each step were recovered and combined. This wash cycle was repeated three times, and supernatants were dried down in a vacuum centrifuge. Samples were reconstituted in deionized water, and oligosaccharides were purified by solid-phase extraction using porous graphitized carbon cartridges. Hypercarb carbon cartridges were conditioned with 3 column volumes of deionized water, followed by 3 volumes of 80% acetonitrile in 0.10% aqueous trifluoroacetic acid (TFA) (v/v) and with another 3 volumes of deionized water. The samples were loaded onto the cartridge, incubated for 10 min at room temperature, washed with 3 volumes of deionized water, eluted with 3 volumes of 40% acetonitrile and 0.1% TFA in water (v/v), and dried in vacuo. Glycans were reconstituted in 5 μL of water. MALDI Fourier transform ion cyclotron resonance (FTICR)-MS was then performed as described previously, using a mixture of maltooligosaccharides as an external calibrant (38). All oligosaccharide compositions were identified by accurate mass, utilizing a 10 ppm tolerance, and five replicate spectra were acquired for each sample. RESULTS
Gel Electrophoretic Analyses of Milk Proteins. Gel electrophoresis was used to determine semiquantitative changes in expression of the major milk proteins. Samples from days 1, 15, and 30 of lactation were examined in these studies. The total protein amount was determined, and 10 μg aliquots were investigated via SDS-PAGE. Shown in Figure 1 are representative SDSPAGE analyses. The 10 abundant proteins indicated were identified using in-gel digestion and peptide mass fingerprinting. The assignments are summarized in Figure 1 and Table 1, along with pertinent identification parameters such as the number of peptides matched (nP), the probability of a stochastic assignment, and the score of the next best protein identification (ΔSM). For reference, a significant match at the 95% CI corresponds to a score of 65. All proteins were identified with scores of 69 or higher under the Mascot protocol. In addition, these results are characterized by large values of ΔSM, which is indicative of a high level of discrimination and a low likelihood of false positive identification afforded by accurate mass analysis (39). To explore the glycosylation status of the abundant milk proteins, gels were run in duplicate and stained with colloidal Coomassie brilliant blue G-250 (for total protein) and Pro-Q Emerald 300 (for glycan content) stains separately. To obtain quantitative information, each gel lane was examined by an inhouse image-processing program. Several proteins were shown to change in total Coomassie signal intensity during lactation. These trends are demonstrated in Figure 2, which shows an overlay of three SDS-PAGE electropherograms generated from a single individual at differing lactation times. Upon examination, it is clear that the expression level of the heavy chain of IgA (immunoglobulin R-1 chain C, IGHA1) is decreasing for this mother over the first lactation month, as expected. In addition to IGHA1, there are four other proteins detailed below whose abundances are found to vary during the first month of lactation.
Figure 1. Coomassie (top) or glycoprotein-specific (bottom) stained SDS-PAGE separations for three mothers at varying time points. Indicated protein bands identified by peptide mass fingerprinting are annotated in Table 1, along with several measures of the quality of the identification. Abbreviations: M, mother code; D, lactation day; and MWM, molecular weight markers. Markers for Coomassie stain are, from top, 203, 120, 95, and 49 kDa. For pro-Q stain, markers are 180, 82, and 42 kDa. Protein K is R-lactalbumin.
All quantitative comparisons of protein and glycan-level variations were made using background-subtracted integrals of Coomassie- or pro-Q-stained electrophoretic peaks, respectively. The dynamic behaviors of five glycoproteins including tenascin, xanthine dehydrogenase (XD), bile salt-stimulated lipase (BSSL), lactoferrin, and IGHA1 protein are shown in Figure 3. These proteins were selected for this comparison because they each signaled under both the Coomassie and the glycoprotein-specific stains. The upper bar graph shows the averaged glycoproteinspecific stain intensity, and the lower graph shows the averaged Coomassie response at days 1, 15, and 30 for each protein. Proteins were identified as differentially glycosylated or expressed if the response to the appropriate stain was significantly different between two time points at greater than the 90% CI. Remarkably, tenascin, IGHA1 protein, XD, and lactoferrin expression levels all decrease significantly from colostrum to mature milk in these
Article
J. Agric. Food Chem., Vol. 58, No. 10, 2010
samples. Interestingly, XD expression peaks at 2 weeks lactation time and drops to approximately half of the peak concentration by 1 month lactation time, while lactoferrin, IGHA1, and tenascin decrease from colostrum to mature milk. Mannose receptor and Ig κ light chain both increase from day 1 to day 30 of lactation (data not shown). Mannose receptor and XD are each potential N-glycoproteins by virtue of the presence of the consensus sequence for N-glycosylation. XD responds to the glycoprotein-specific stain used in this study. There is no corresponding response from the mannose receptor. Interestingly, there are also glycoproteins that exhibit disparate behavior under protein- or glycan-specific staining. A small section of the SDS-PAGE gel band is shown for illustrative purposes in Figure 4, with samples from the first 10 days of lactation shown. This detailed examination was intended to determine if any interesting dynamic behavior was missed between days 1 and 15 as examined previously. The CBB stain intensities suggest that the gross quantity of lactoferrin remained relatively constant over the first 10 days of lactation. Surprisingly, the total glycan stain intensity decreases by approximately 60% over that time. By day 15, this decrease in glycosylation is restored to near its previous levels. As one of the most abundant glycoproteins in milk, this trend represents a significant change in the total glycoconjugate content present in the infant’s digestive system. Additionally, while the expression level of XD peaks at
6443
2 weeks and decreases afterward (days 15 and 30 are different at the 95% CI), the glycosylation level is largely unchanged from 2 to 4 weeks, suggesting an increase in the degree of glycosylation of this glycoprotein during this time. Although the CBB and glycan-specific staining methods have previously both been shown to be valid quantitative platforms (40,41), the changes in lactoferrin glycosylation were also verified
Table 1. Summary of All Proteins Identified by Peptide Mass Fingerprintinga gel band A B C D E F G H I J
protein identity tenascin fatty acid synthase mannose receptor xanthine dehydrogenase bile salt-stimulated lipase lactoferrin serum albumin Ig R-1 chain C κ-casein Ig κ light chain
a
MW (kDa) SM ΔSM nP SC (%) erms(ppm) 240.6 275.5 168.9 148.4 66.6 79.8 67.7 54.3 20.4 23.5
163 143 93 80 94 97 178 73 75 71
135 111 61 42 71 69 147 32 37 N/A
30 30 19 10 12 15 22 6 5 5
24 20 19 12 25 29 48 20 45 41
3 2 4 4 2 3 1 2 1 2
Several parameters relating to the quality of the identification are also tabulated as follows: SM, Mowse score; ΔSM, difference between the top two highest scoring protein identifications; np, number of peptides identified; SC (%); percent sequence coverage of the protein identified; and eRMS (ppm), root mean square mass error of identified peptides, in parts per million. The identification for Ig κ chain C has no ΔSM listed because all other proteins identified at a nonstochastic level were splice variants.
Figure 3. Glycosylation (upper) or expression (lower) levels of selected human milk glycoproteins. Singly, doubly, or triply marked entries are statistically different at the 90, 95, or 99% CI from the corresponding day 1 entry. Standard deviations for each measurement are shown as error bars. When necessary, signals were scaled by the factor in parentheses to enable direct comparison between highly and moderately abundant glycoproteins.
Figure 2. Electropherograms of SDS-PAGE-separated, Coomassie-stained proteins with annotated identifications as detailed in Table 1. Protein K is R-lactalbumin.
6444
J. Agric. Food Chem., Vol. 58, No. 10, 2010
Froehlich et al.
Figure 4. Image line profiles of isolated milk proteins stained with either Coomassie (left) or Pro-Q (right) stains. Proteins were isolated from a single mother on lactation days 1, 2, 5, and 10. While lactoferrin is shown to have a relatively constant total protein level (top left), the degree of glycosylation at day 10 is approximately 40% of that present at day 1 (top right). The lower two traces showing Ig R-1 chain C behavior show little variation.
by another method. Enzymatic release of N-glycans followed by analysis by MS was used both to verify the gross glycosylation trends that were observed via the glycan-specific visualization and to identify the compositions of the N-glycans. Shown in Figure 5 are the mass spectra obtained from enzymatic release of electrophoretically separated lactoferrin from different lactation days. The glycosylation trends obtained from the Pro-Q Emerald 300 staining approximate the total glycan intensity changes observed via high-resolution, high mass accuracy FTICR-MS. The glycans observed were primarily biantennary, complex type, and fucosylated, and compositions for each peak are labeled on the spectra. A decrease in both the total level of glycosylation and the degree of fucosylation was observed between days 2 and 5. Shotgun Proteomics Analysis of Milk Proteins. LC-MS/MS analyses were performed to determine the gross protein and glycoprotein content in milk. As expected, the shotgun analyses identified significantly more proteins than the gel-based analyses. The LC-MS/MS analyses identified a total of 55 proteins present in 11 samples analyzed. Identified proteins were searched in Swiss-Prot to determine their annotated glycosylation status. Shown in Table 2 are the proteins identified along with their potential for glycosylation. Of the 55 proteins identified, 27 are
glycosylated, and an additional 12 are potentially N-glycosylated based on the presence of the consensus sequence NXS or NXT, where X is any amino acid except proline. O-Glycosylation can occur at essentially any serine or threonine residue, so there is no analogous classification for potential O-glycosylated proteins, as essentially no filter exists based on amino acid sequence. DISCUSSION
While there are a small number of published studies (
